Mullerian Inhibiting Substance (MIS) is produced by the fetal testis as a 140 kDa glycosylated disulfide-linked homodimer that causes regression of the Mullerian duct in the male fetus. Under reducing conditions, the protein migrates on gel electrophoresis at an apparent molecular weight of 70 kDa. The protein can be proteolytically cleaved by exogenous plasmin into two distinct fragments that migrate electrophoretically as 57 kDa and 12.5 kDa moieties with cleavage at residue 427 of the intact 535 amino acid monomer (Pepinsky, et al., J. Biol. Chem. 263:18961-4 (1988)).
Various methods for purifying MIS are known. U.S. Pat. No. 4,404,188, filed Jul. 29, 1981 and entitled "Purified Mullerian Inhibiting Substance and Method of Purification" describes a process for purifying MIS which comprises treatment with a protein inhibitor, chromatography on ion exchange, chromatography on wheat germ lectin, on concanavalin A and/or on a supported triazinyl dye. U.S. Pat. No. 4,487,833, filed on Mar. 1, 1982 and entitled "Method of Preparing Hybridomas and of Purifying Immunogenic Materials" describes a process for separating MIS using immunoaffinity chromatography. U.S. Pat. No. 5,011,687, filed Oct. 19, 1985 entitled "Purified Mullerian Inhibiting Substance and Process for Treating Human Ovarian Cancer Cells," describes a process for purifying MIS from testes by using aqueous polar dissociative solutions, separation of DNA and RNA, fractionation by gel filtration chromatographic elution, and isolation of the MIS. MIS may also be obtained from recombinant DNA techniques (Cate et al., Cell 45:685-698 (1986); Cate et al., U.S. Pat. No. 5,047,336).
In the female fetus, the Mullerian duct develops into the Fallopian tubes, uterus and upper vagina. It is known that MIS causes regression of the Mullerian duct in the male fetus. MIS has also been shown to play a role in inhibition of oocyte meiosis (Takahashi et al., Mol. Cell. Endocrinol. 47:225-234 (1986)), testicular descent (Hudson et al., Endocr. Rev. 7:270-283 (1986)), inhibition of fetal lung development (Catlin et al., Am. J. Obstet. Gynecol. 159:1299-1303 (1988)), inhibition of autophosphorylation of the EGF receptor (Coughlin et al., Mol. Cell. Endocrinol. 49:75-86 (1987); Cigarroa et al., Growth Factors 1:179-191 (1989)) and inhibition of tumor growth (Donahoe et al., Science 205:913-915 (1979); Donahoe et al., Ann. Surg. 194:472-480 (1981); Fuller, Jr., et al., J. Clin. Endocrinol. Metab. 54:1051-1055 (1982); Fuller, Jr. et al., Gynecol. Oncol. 22:135-148 (1985); U.S. Pat. No. 4,404,188, Donahoe, P. K., et al., filed Jul. 29, 1981).
The antitumor effect of MIS demonstrated in U.S. Pat. No. 5,011,687, was elicited using natural MIS extracted and partially purified from bovine testes. Since that time, the bovine, human, and mouse MIS genes have been cloned and the human protein expressed in Chinese hamster ovary (CHO) cells. Initial antiproliferative studies against established cell lines involved using a highly purified recombinant human MIS (rhMiS), but these studies suggested that the antiproliferative effect of rhMIS on human gynecological tumor cells is limited to ovarian cancers. (Wallen et al., Cancer Res. 49:2005-2011 (1986)). Furthermore, tumor response to the highly purified rhMIS was inconsistent and preparation dependent (Wallen et al., Cancer Res. 49:2005-2011 (1986)).
As stated, digestion of MIS with plasmin cleaves at a site 109 amino acids from the carboxy-terminus producing a 25-kDa fragment and a high molecular mass complex derived from the amino terminus of the protein. However, the N- and C-terminal fragments remain associated as a non-covalent complex. In Pepinsky et al., Journal Biol. Chem. 263:18961-18964 (1988), proteolysis of MIS with plasmin into N- and C-terminal fragments did not alter MIS activity in the organ culture assay measuring the ability of MIS to promote regression of the Mullerian duct. However, attempts to dissociate the non-covalent complex by acidifying the sample with acetic acid or after boiling, destroyed MIS activity in the organ culture assay. In contrast, when the two fragments were dissociated with 1% sodium deoxycholate, MIS activity in the organ culture assay was not altered. However, Pepinsky et al. were unable to determine whether both fragments were necessary for biological activity.
A later review article on MIS, Cate et al., Mullerian-Inhibiting Substance, in "Handbook of Experimental Pharmacology" 95/11:179-210 (1990), suggests that both the N- and C-terminal domains are necessary for regression of the Mullerian duct. The authors found that the N- and C-terminal fragment dimers were inactive when assayed individually in the organ culture assay. However, when incubated together, regression of the Mullerian duct was observed.
Recently, gene therapy has become a viable method for treating tumors. Tumor-infiltrating-lymphocytes (TILs) have been shown efficacious in the treatment of metastatic tumors in mice and man when administered with inserted genes encoding chemotherapeutic agents. Exogenous genes can be inserted into TILs in vitro and then reinjected into a patient (Rosenberg et al., Science 233:1318-21 (1986); Spiess et al., J Natl Cancer Inst 79:1067-75 (1987); Cameron et al., J Exp Med 171:249-63 (1990); Rosenberg et al., N. Engl. J. Med. 319:1676-80 (1988); Alexander et al., J of Immunotherapy 10:389-97 (1991); Culver et al., PNAS U.S.A. 88:3155-3159 (1991); Blaese et al., Clin. Res. 37(2):599A (1989); Kasid et al., PNAS U.S.A. 87:473-77 (1990)). Direct in situ introduction of exogenous genes into proliferating tumors has also been described (Culver et al., Science 256:1550-2 (1992); Ram et al., Can. Res. 53:83-88 (1991)).
The major histocompatibility complex is a group of closely linked genes which encode molecules that restrict the specificity of antigen recognition by T lymphocytes. The antigens fall into two classes: class I and class II. The immunological aspects of the present invention are reviewed, for example, by Klein, J., In: Immunology: The Science of Self-Non-Self Discrimination, Wiley-Interscience, NY, pp. 270-309 (1982) and Feldman, M. et al., Scientific American 259:60-85 (1988), which references are herein incorporated by reference. Antigens of class I restrict antigen recognition predominantly by cytotoxic T lymphocytes, whereas antigens of class II restrict recognition by regulatory T cells. Class I MHC molecules are expressed on all cells. In contrast, MHC class II molecules are expressed predominantly, on B lymphocytes.
Agents capable of modulating the expression of the MHC class I antigens may be employed in the treatment or prevention of metastatic cancer, immunodeficiency diseases, and organ and tissue rejection.